Protein translocation is a highly conserved process that is essential to all life. Extracellular secretion of virulence factors is a strategy utilized by invading bacteria to establish a colonization niche, communicate with host cells, and modulate host defense and response. With few exceptions, bacterial protein secretion systems are characterized by the membrane translocation of a single protein or else small protein complexes (Christie et al., “Bacterial Type IV Secretion: Conjugation Systems Adapted to Deliver Effector Molecules to Host Cells,” Trends Microbiol 8:354-60 (2000); Galan et al., “Type III Secretion Machines: Bacterial Devices for Protein Delivery into Host Cells,” Science 284:1322-8 (1999); Gentschev et al., “The E. coli alpha-Hemolysin Secretion System and its Use in Vaccine Development,” Trends Microbiol 10:39-45 (2002); Henderson et al., “Autotransporter Proteins, Evolution and Redefining Protein Secretion,” Trends Microbiol 8:529-32 (2000); and Russel M., “Macromolecular Assembly and Secretion Across the Bacterial Cell Envelope: Type II Protein Secretion Systems,” J Mol Biol 279:485-99 (1998)). Recently, however, the production and release of outer membrane vesicles (OMVs) has been demonstrated as a novel secretion mechanism for the transmission of a diverse group of proteins and lipids to mammalian cells (Kuehn M. J. et al., “Bacterial Outer Membrane Vesicles and the Host-Pathogen Interaction,” Genes Dev 19:2645-55 (2005)). OMVs are small proteoliposomes with an average diameter of 50-200 nm that are constitutively released from the outer membrane of pathogenic and non-pathogenic species of Gram-negative bacteria during growth (Beveridge T. J., “Structures of Gram-Negative Cell Walls and their Derived Membrane Vesicles,” J Bacteriol 181:4725-33 (1999)). Biochemical analysis has demonstrated that OMVs are comprised of outer membrane proteins, lipopolysaccharide, phospholipids and soluble periplasmic proteins, (Horstman et al., “Enterotoxigenic Escherichia coli Secretes Active Heat-Labile Enterotoxin Via Outer Membrane Vesicles,” J Biol Chem 275:12489-96 (2000) and McBroom et al., “Outer Membrane Vesicles,” In EcoSal—Escherichia coli and Salmonella: Cellular and Molecular Biology (III, R. C., ed.). ASM Press, Washington, D.C. (2005)) the latter of which become trapped in the vesicle lumen during release from the cell surface. OMVs are largely devoid of inner membrane and cytoplasm components although several studies indicate that chromosomal, phage and plasmid DNA can infiltrate OMVs as a means of OMV-mediated transfer of genetic information between bacteria (Dorward et al., “Export and Intercellular Transfer of DNA Via Membrane Blebs of Neisseria gonorrhoeae,” J Bacteriol 171:2499-505 (1989); Kolling et al., “Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli 0157:H7,” Appl Environ Microbiol 65:1843-8 (1999); Yaron et al., “Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli 0157:H7 to Other Enteric Bacteria,” Appl Environ Microbiol 66:4414-20 (2000); and Renelli et al., “DNA-Containing Membrane Vesicles of Pseudomonas aeruginosa PAO1 and their Genetic Transformation Potential,” Microbiology 150:2161-9 (2004)).
An intriguing yet poorly understood phenomena pertaining to OMVs is the observation that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including, for example, Escherichia coli cytolysin A (ClyA), (Wai et al., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003)) enterotoxigenic E. coli heat labile enterotoxin (LT), (Horstman et al., “Enterotoxigenic Escherichia coli Secretes Active Heat-Labile Enterotoxin Via Outer Membrane Vesicles,” J Biol Chem 275:12489-96 (2000)) and Actinobacillus actinomycetemcomitans leukotoxin, (Kato et al., “Outer Membrane-Like Vesicles Secreted by Actinobacillus actinomycetemcomitans are Enriched in Leukotoxin,” Microb Pathog 32:1-13. (2002)) whereas proteins that are excluded from OMVs include numerous unidentified outer membrane (OM) proteins (Kato et al., “Outer Membrane-Like Vesicles Secreted by Actinobacillus actinomycetemcomitans are Enriched in Leukotoxin,” Microb Pathog 32:1-13. (2002)) as well as E. coli DsbA (Wai et al., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003)). The preferential exclusion of proteins raises the interesting possibility that a yet-to-be determined sorting mechanism exists in the bacterial periplasm for discriminatory loading of a highly specific subset of proteins into OMVs (Wai et al., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003) and McBroom et al., “Release of Outer Membrane Vesicles by Gram-Negative Bacteria is a Novel Envelope Stress Response,” Mol Microbiol 63:545-58 (2007)). Moreover, the observation that certain virulence factors are enriched in vesicles suggests that OMVs may play a key role in bacterial pathogenesis by mediating transmission of active virulence factors and other bacterial envelope components to host cells. Indeed, numerous vesicle-associated virulence factors (e.g., adhesins, immunomodulatory compounds, proteases and toxins) have been shown to induce cytotoxicity, confer vesicle binding to and invasion of host cells, and modulate the host immune response (Horstman et al., “Enterotoxigenic Escherichia coli Secretes Active Heat-Labile Enterotoxin Via Outer Membrane Vesicles,” J Biol Chem 275:12489-96 (2000); Fiocca et al., “Release of Helicobacter pylori Vacuolating Cytotoxin by Both a Specific Secretion Pathway and Budding of Outer Membrane Vesicles. Uptake of Released Toxin and Vesicles by Gastric Epithelium,” J Pathol 188:220-6 (1999); Keenan et al., “A Role for the Bacterial Outer Membrane in the Pathogenesis of Helicobacter pylori Infection,” FEMS Microbiol Lett 182:259-64 (2000); Kadurugamuwa et al., “Delivery of the Non-Membrane-Permeative Antibiotic Gentamicin into Mammalian Cells by Using Shigella flexneri Membrane Vesicles,” Antimicrob Agents Chemother 42:1476-83 (1998); and Kesty et al., “Enterotoxigenic Escherichia coli Vesicles Target Toxin Delivery into Mammalian Cells,” EMBO J 23:4538-49 (2004)).
To date, one of the best studied vesicle-associated virulence factors is the 34-kDa cytotoxin ClyA (also called HlyE or SheA) found in pathogenic and non-pathogenic E. coli strains (Wai et al., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003) and del Castillo et al., “The Escherichia coli K-12 SheA Gene Encodes a 34-kDa Secreted Haemolysin,” Mol Microbiol 25:107-15 (1997)) and also in Salmonella enterica serovars Typhi and Paratyphi A (Oscarsson et al., “Characterization of a Pore-Forming Cytotoxin Expressed by Salmonella enterica serovars typhi and paratyphi A,” Infect Immun 70:5759-69 (2002)). Structural studies indicate that the water-soluble form of ClyA is a bundle of four major α-helices, with a small surface-exposed hydrophobic beta-hairpin at the “head” end of the structure, and the N- and C-termini at the “tail” end (Wallace et al., “E. coli Hemolysin E (HlyE, ClyA, SheA): X-ray Crystal Structure of the Toxin and Observation of Membrane Pores by Electron Microscopy,” Cell 100:265-76 (2000)) while lipid-associated ClyA forms an oligomeric pore complex comprised of either 8 or 13 ClyA subunits (Eifler et al., “Cytotoxin ClyA from Escherichia coli Assembles to a 13-meric Pore Independent of its Redox-State,” EMBO J 25:2652-61 (2006) and Tzokov et al., “Structure of the Hemolysin E (HlyE, ClyA, SheA) Channel in its Membrane-Bound Form,” J Biol Chem 281:23042-9 (2006)). Expression of the clyA gene is silenced in non-pathogenic E. coli K-12 laboratory strains by the nucleoid protein H-NS (Westermark et al., “Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli,” J Bacteriol 182:6347-57 (2000)) but is derepressed in H-NS-deficient E. coli, thereby inducing cytotoxicity towards cultured mammalian cells (Gomez-Gomez et al., “Hns Mutant Unveils the Presence of a Latent Haemolytic Activity in Escherichia coli K-12,” Mol Microbiol 19:909-10 (1996)). More recent evidence indicates that ClyA is exported from E. coli in OMVs and retains a cytolytically active, oligomeric conformation in the vesicles (Wai et al., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003)). However, the route by which ClyA manages to cross the bacterial IM and assemble in OMVs remains a mystery, as it carries no canonical signal peptide (del Castillo et al., “The Escherichia coli K-12 SheA Gene Encodes a 34-kDa Secreted Haemolysin,” Mol Microbiol 25:107-15 (1997)) and is not N-terminally processed (Ludwig et al., “Analysis of the SlyA-Controlled Expression, Subcellular Localization and Pore-Forming Activity of a 34 kDa Haemolysin (ClyA) from Escherichia coli K-12,” Mol Microbiol 31:557-67 (1999)). Also undetermined is the role that ClyA plays in vesicle-mediated interactions with mammalian cells.
The present invention is directed to overcoming these and other deficiencies in the art.